SUMMARY Swelling caused by edematous fluid retention is a common, life-threatening symptom of heart failure (HF) and chronic kidney disease (CKD). Loop diuretics are often prescribed as a first-line therapy to quickly reduce the extracellular fluid volume burden in HF and CKD patients. This class of diuretic works by inhibiting NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop, and increases the delivery of NaCl and fluid to the distal nephron comprised of the distal convoluted tubule (DCT) and collecting duct (CD). In response to the increased NaCl and fluid load, the DCT and CD increase their NaCl reabsorbing capacity by upregulating the expression of specific ion transporters and channels. This compensatory mechanism diminishes the effectiveness of loop diuretics and gives rise to loop diuretic resistance. A growing consensus among nephrologists is that distally acting diuretics that inhibit sodium (Na+) reabsorption in the DCT (i.e. thiazide diuretics) or CD (potassium-sparing diuretics) downstream of the TAL should be administered in an effort to overcome loop diuretic resistance. However, both diuretic classes have critical limitations that highlight the need to discover more effective, safer, and novel-mechanism distal diuretics for circumventing loop diuretic resistance. In this application, we propose to discover the first potent and selective inhibitors of heteromeric Kir4.1/5.1 potassium channels, which have emerged recently as key regulators of NaCl reabsorption and kinase signaling in the distal nephron. In Aim 1, we will employ a fully validated, fluorescence-based thallium-flux assay to screen approximately 110,000 structurally diverse compounds from the Vanderbilt Institute of Chemical Biology library for novel inhibitors of Kir4.1/5.1 channels heterologously expressed in HEK-293 cells. A series of secondary thallium-flux assays, as well as high-throughput automated patch clamp electrophysiology, will then be used to evaluate the potency and selectivity of confirmed inhibitors for Kir4.1/5.1 over an extensive panel of related inward rectifier potassium (Kir) channels. In Aim 2, we will select the most promising Kir4.1/5.1 inhibitors based on their potency, selectivity, chemical structure, and in vitro metabolic stability properties to develop analog libraries using state-of-the-art medicinal chemistry techniques with the goal of optimizing the pharmacological properties of inhibitors for in vivo administration. In Aim 3, we will use single channel analysis to test the activity lead inhibitors against native rat and human Kir4.1/5.1 channels in freshly isolated kidney tubules. In addition, we will test the hypothesis that inhibition of Kir4.1/5.1 induces renal excretion of Na+, K+, and water in rats. Completion of these aims will provide critically needed tool compounds for evaluating the therapeutic potential of Kir4.1/5.1 channels as a diuretic target in the setting of loop diuretic resistance in HF and CKD patients.